Composition For The Prevention And Treatment Of Influenza Virus Infection And Composition For Suppressing Neuraminidase Activity Comprising Turmeric Extract

ABSTRACT

Disclosed is a composition comprising a turmeric extract for preventing and treating influenza virus infection and for inhibiting neuraminidase activity. A turmeric extract, its fraction, and a curcuminoid-based compound separated therefrom may inhibit neuraminidase activity and have antiviral and cell degeneration inhibitory effects on influenza virus, and may be useful in preventing and treating influenza virus infection.

BACKGROUND

1. Field

The present invention relates to a composition comprising a turmericextract for the prevention and treatment of influenza virus infectionand the inhibition of neuraminidase activity.

2. Description of Related Art

Influenza virus is one of the most infective viruses that cause acuterespiratory diseases, and in severe instances, cause herd infection orpandemic over the world, particularly giving rise to serious respiratorysymptoms in children, the aged, patients with cardiopulmonary diseases,and the like (Hien, T. T. et al. N. Eng. J. Med., 350, 1179, 2004).Influenza virus is a genus of the Orthomyxovirus and has three types,that is, A, B, and C. Among them, particularly A and B types routinelyspread in people. Influenza virus has eight RNA gene segments, and twoglycoproteins, hemagglutinin (HA) and neuraminidase (NA) that are a typeof surface antigen found on the surface of the influenza virus.Hemagglutinin is a trimer with a head and a stalk, and the head of thetrimer involves a majority of antigenic variation. Hemagglutinin bindsto terminal sialic acid residues on the surface of host cells to enableviruses to attach to and enter in the host cells in sequence(Chandrasekaran, A. et al. Nature biotechnology 26, 107, 2008).Neuraminidase is a mushroom-shaped tetramer with a head and a stalk, andthe head of the tetramer has active sites on the top surfaces thereof.Neuraminidase cleaves the alpha-ketosidic bond between oligosacchridesat cell surfaces and terminal neuraminic acid residues to assist virusesreplicated and proliferated in the infected cells to exit outside thehost cells and enter in the respiratory mucosa cells (a. Mark, V.I.Nature review 6, 967, 2007. b. Huberman, K. et al. Virology 214, 294,1995).

The same subtype of virus surface antigen produces an antigenicvariation to generate new antigenic variants every year. Particularly,among influenza viruses, avian influenza virus, which is still a threatundergoes an antigenic shift to infect various kinds of birds such aschickens, turkeys, ducks, wild birds, and the like. Avian influenzavirus spreads so quickly that once a chicken is infected, 80% or more ofthe chickens are killed. Avian influenza virus which poses the mostdamage and threat to the poultry industry over the world causes viraldiseases, and this pervasive effect is not just limited to the poultryindustry. It has been reported that avian influenza virus can infecthumans, which causes diseases to spread among humans (Gubareva, L. V. etal. Lancet. 355, 2000). In past ages, influenza virus has been known tocause many diseases. Specifically, there have been three flu pandemicsin the twentieth century, Spanish flu pandemic (H1N1) which killed aboutthirty millions, the Asian Flu (H2N2) which killed and an estimated onemillion and the Hong Kong Flu (H3N2) which also killed an estimated onemillion. Later, in 2003 to 2008, 385 were infected and 243 died.Recently, the World Health Organization (WHO) officially announced thatthe novel swine-origin influenza break out of April, 2009 as beingpandemic. As of Jun. 29, 2009, 70,893 people (including 311 deaths) inover 115 countries were infected, and a South Korean nun visiting Mexicofor volunteer work has been confirmed as the first flu patient on May 2,2009. As of Aug. 16, 2009, a cumulative number of 2,089 flu patients(including 2deaths) were reported in South Korea.

To prevent and treat the influenza virus infection, consideration may bemade to inhibit the absorption in epithelial cells, the invasion intocells, the transcription or replication of genes, the synthesis ofproteins, or the release from cells, each having been the focus of theantiviral studies.

To treat diseases caused by influenza virus, four substances, that is,Amatadine, Rimatadine, Zanamivir, and Oseltamivir have been used withthe approval of Food and Drug Administration (FDA). As older M2inhibitors, Amatadine and Rimatadine have antiviral effects by blockingan ion channel of a membrane protein, particularly M2 protein that isessential to the proliferation of virus to inhibit the uncoating of thevirus, but are only effective against influenza A virus. Also, it isreported that the virus becomes more tolerant and resistant to thesubstances as a consequence of being used over 40 years, and severe sideeffects occur in the nervous system and stomach (Bantia, S. et al.Antiviral Research 69, 39, 2006). Since 1999, as new drugs to treatvirus infection, Zanamivir and Oseltamivir called neuraminidaseinhibitors have been used, which play an important role in proliferationof virus, have a low prevalence of tolerance, and are active againstboth influenza A and B viruses (Zhang, J. et al. Bioorg. Med. Chem.Lett. 16, 3009, 2006).

However, Zanamivir has an advantage of high antiviral effects but isdisadvantageous in low bioavailability and quick release from thekidney, and Oseltamivir causes severe vomiting (Ryan, D. M. et al.Antimicrob. Agents Chemother., 39, 2583, 1995).

As mentioned above, existing antivirals have serious side effects andrequire considerable caution in their application. Also, the developmenteffects of vaccines are low when the vaccine virus is not matched tocirculating viruses. Accordingly, there is an increasing need for a newinfluenza antiviral with excellent infection inhibition and stability.

To satisfy the need, the inventors invented the present invention afterdiscovering that a turmeric extract, its fraction or a curcuminoid-basedcompound separated therefrom has neuraminidase inhibitory activity, andantiviral and cell degeneration inhibitory effects.

DISCLOSURE

It is an object of the present invention to provide a pharmaceuticalcomposition for preventing and treating influenza virus infection and atreatment method using the same.

It is another object of the present invention to provide a foodcomposition for preventing and reducing influenza virus infection.

It is yet another object of the present invention to provide aquasi-drug composition for preventing and reducing influenza virusinfection. It is still another object of the present invention toprovide a neuraminidase inhibitor composition for inhibiting theactivity of neuraminidase present in various biospecies to treatdiseases caused thereby, and an inhibition method using the same.

EFFECT OF THE INVENTION

The composition comprising the turmeric extract, fraction, andcurcuminoid-based compound separated therefrom according to the presentinvention may inhibit neuraminidase activity and have antiviral and celldegeneration inhibitory effects, and thus may be useful in preventingand treating influenza virus infection.

BEST MODE

To achieve this object, in an aspect of the present invention, acomposition comprising a turmeric extract or its fraction as an activeingredient to prevent and treat influenza virus infection is provided.

In another aspect of the present invention, a composition comprising acompound represented by the following chemical formula 1 as an activeingredient to prevent and treat influenza virus infection is provided:

where R is independently hydrogen, a hydroxyl group, or C₁-C₁₀ alkoxygroup.

In another aspect of the present invention, a use of a turmeric extract,a turmeric fraction, or a compound represented by the following chemicalformula 1 in preventing and treating influenza virus infection, or a useof a turmeric extract, a turmeric fraction, or a compound represented bythe following chemical formula 1 in preparing a drug for preventing andtreating influenza virus infection is provided.

In another aspect of the present invention, a method for treatinginfluenza virus infection is provided, including administering, to anindividual infected or to be infected with influenza virus, acomposition comprising a turmeric extract, a turmeric fraction, or acompound represented by the following chemical formula 1 as an activeingredient to prevent and treat influenza virus infection:

where R is independently hydrogen, a hydroxyl group, or C₁-C₁₀ alkoxygroup.

In another aspect of the present invention, a composition comprising aturmeric extract, a turmeric fraction, or a compound represented by thefollowing chemical formula 1 as an active ingredient to inhibitneuraminidase activity is provided:

where R is independently hydrogen, a hydroxyl group, or C₁-C₁₀ alkoxygroup.

In another aspect of the present invention, a method for inhibitingneuraminidase activity including contacting the composition with asample containing neuraminidase is provided.

Hereinafter, the present invention is described in more detail.

The present invention provides a composition comprising a turmericextract or its fraction as an active ingredient to prevent and treatinfluenza virus infection.

The composition of the present invention may inhibit the activity ofneuraminidase that is found on the surface of the influenza virus andplays an essential role in replicating the virus, and may prevent theinfluenza virus from spreading to other cells in the respiratory organ,thereby reducing, preventing, and treating influenza virus infection.

The term “prevent” described herein refers to all activities to inhibitthe influenza virus infection or retard the pathogenesis of theinfluenza virus infection by administering the composition.

The term “treat” described herein refers to all activities to improve orfavorably change the symptoms of the influenza virus infection byadministering the composition.

The influenza virus refers to a strain of influenza virus that may causediseases to human or animal individuals, and may be influenza A virus,influenza B virus, or influenza C virus which are identified bydifferences in nucleoprotein and matrix protein. The influenza A virus,influenza B virus, and influenza C virus may include their variants. Theinfluenza virus subtypes are mainly classified on the basis ofcharacteristics of hemaglutinin (HA) and neuraminidase (NA), and todate, 6 types of HAs (H1-H6) and 9 types of NAs (N-1-N9) have beenreported. Preferably, the influenza virus may be H1N1 influenza virus orH9N2 influenza virus, more preferably rvH1N1 influenza A virus, H1N1influenza virus (A/PR/8/34), or H9N2 influenza virus(A/Chicken/Korea/MS96/96). The influenza virus may cause influenza,cold, laryngopharyngitis, bronchitis, pneumonia, and particularly, birdflu, swine flu, or goat flu.

Generally, a turmeric is a tuberous root of Curcuma longa Linne as it isor a tuberous root of Curcuma longa Linne steamed and dried afterremoving the periderm (the Korean Pharmacopoeia 9^(th) Edition), and the“tuberous root” called a storage root is a thickened root.

In the present invention, a turmeric refers to a tuberous root of aplant of Curcuma sp. including, but not limited to, Curcuma wenyujin (Y.H. Chen et C. Ling), Curcuma longa Linne, Curcuma longa Salisb., Curcumazedoaria, Curcuma kwansiensis (S. G. Lee et C. F. Liang), Curcumaaeruginosa, Curcuma phaeocaulis Val. (Zingiberaceae), and Curcumadomestica.

For example, Curcuma longa Linne has a rhizome by Curcuma longa Rhizoma(the Korean Pharmacopoeia 9^(th) Edition), and the “rhizome” called arootstalk refers to a stem of a plant creeping underground like a root.

Plants of Curcuma sp. have a “tuberous root” and a “rhizome”.

Among plants of Curcuma sp., the measurement results of the content of acurcumin derivative in an ethanol extract of Curcuma longa Linne forarea showed that 17.0 g/kg of curcumin, 5.3 g/kg of demethoxycurcumin,and 3.4 g/kg of bisdemethoxycurcumin were detected in a tuberous root,these compounds were not detected in a stem, and 2.8 g/kg of curcumin,0.4 g/kg of demethoxycurcumin, and 6.8 g/kg of bisdemethoxycurcumin weredetected in a rhizome (Example 4).

In the present invention, a turmeric extract may be obtained fromdifferent organs of native plants, hybrids, or varieties, preferably atuberous root or a plant tissue culture.

In the present invention, a turmeric may be available on the market, maybe collected in nature, or may be obtained from cultivated products.

In another aspect of the present invention, a method for preparing acomposition having activity against neuraminidase is provided, includingextracting a turmeric with a solvent of water, C₁-C₄ alcohol, ormixtures thereof to obtain a turmeric extract.

Also, the method may further comprise fractioning the turmeric extractwith hexane, ethylacetate, or water.

Also, the method may include separating and purifying the turmericfraction.

The obtaining of the turmeric extract may use conventional extractiontechniques in the art, including, but not limited to, for exampleultrasonic extraction, maceration, heat extraction, cold extraction,filtration, reflux extraction, and the like. The extraction may beperformed at room temperature or by heating under such conditions as toeliminate or minimize the destruction of an active ingredient. Also, theextraction may use a variety of solvents known in the art, including,but not limited to, for example water, C₁-C₄ alcohol, acetone,ethylacetate, chloroform, or mixtures thereof. Preferably, the turmericextract may be obtained by washing a turmeric root to remove animpurity, drying, milling, and extracting with a solvent of water, C₁-C₄alcohol, or mixtures thereof, more preferably C₁-C₄ alcohol, mostpreferably methanol or ethanol. In this instance, the content of theextractant, that is, the solvent used may be preferably twice to twentytimes as much the dried weight of the turmeric. For example, the alcoholextract may be obtained by drying the turmeric, cutting fine, putting inan extract container, adding a solvent of lower C₁-C₄ alcohol ormixtures thereof, preferably methanol or ethanol, placing at roomtemperature for a predetermined time, and filtering. In this instance,the predetermined time is preferably a week, and concentration orfreeze-drying may be further performed.

The extract of the present invention may include at least one of anextract obtained by extraction, a diluent or a concentrate of anextract, a dried extract obtained by drying an extract, and a productobtained by crudely purifying or purifying an extract.

The turmeric fraction of the present invention may be obtained byfractioning the turmeric extract. To obtain the turmeric fraction, avariety of solvents known in the art may be used. For example, thesolvents may include pentane, hexane, 2,2,4-trimethylpentane, dicainum,cyclohexane, carbon disulfide, carbon tetrachloride, chlorobutane,diisopropylether, chloroform, acetone, nitropropane, butanone,dichloroethane, pyridine, propanol, methanol, ethylacetate, and butanol.More specifically, the turmeric fraction may be obtained by suspendingthe turmeric extract in water, and sequentially fractioning with hexaneand ethylacetate to yield a hexane fraction, an ethylacetate fraction,and a water fraction.

Also, the present invention provides a composition comprising a compoundrepresented by the following chemical formula 1 as an active ingredientto prevent and treat influenza virus infection:

where R is independently hydrogen, a hydroxyl group, or C₁-C₁₀ alkoxygroup.

In this instance, the compound represented by chemical formula 1 may beseparated from the turmeric extract or turmeric fraction. Morespecifically, the compound represented by chemical formula 1 may be acompound represented by any one of the following chemical formulas 2 to4.

Also, the present invention provides a method for separating thecompound represented by chemical formula 1 from a turmeric, including a)extracting the turmeric with a solvent of water, C₁-C₄ alcohol, ormixtures thereof to yield a turmeric extract, b) obtaining a hexanefraction, an ethylacetate fraction, or a water fraction from theextract, and c) separating and purifying the fraction.

More specifically, the compound represented by chemical formula 1according to the present invention may be a representative activeingredient present in a turmeric, and a method for separating thecompound may be performed below.

First, a turmeric may be extracted with a solvent of water, C₁-C₄alcohol, or mixtures thereof to obtain a turmeric extract (step 1). Theturmeric is not particularly limited, and may be available on the marketor from cultivated products. The turmeric may be used after washing anddrying. The alcohol may be lower alcohol such as methanol, ethanol,propanol, or butanol, preferably methanol or ethanol.

In step 1, the turmeric extract may be obtained by drying a turmericunder the shade, cutting fine, milling to improve the extractionefficiency, putting in an extract container, adding a proper amount ofalcohol, placing at room temperature for 5 days, and filtering through afilter paper and the like. This process may be repeated several times,and thereafter concentration or freeze-drying may be further performed.

Subsequently, the turmeric extract obtained in step 1 may be suspendedin water and sequentially fractioned with hexane and ethylacetate toobtain a turmeric fraction (step 2). In this instance, typicalfractional extraction may be used, preferably using a separatory funnel.The turmeric fraction may be a hexane fraction, an ethylacetatefraction, or a water fraction.

Next, the turmeric fraction obtained in step 2 may be separated bysilica gel chromatography, followed by purification, to obtain thecompound represented by chemical formula 1 (step 3). When silica gelchromatography is used for separation, solvents of n-hexane,n-hexane/ethylacetate, chloroform/acetone, and methanol may bepreferably used as mobile phases. Also, a mixed solvent ofn-hexane/acetone may be additionally used. In this instance, a ratio byvolume of n-hexane and ethylacetate may be preferably 50:1 to 1:5, and aratio by volume of chloroform and acetone may be preferably 150:1 to1:4. The chromatography may be performed 1 to several times until asingle compound is obtained, and when needed, concentration orre-crystallization may be performed.

Also, the compound represented by chemical formula 1 according to thepresent invention may be used in the form of a pharmaceuticallyacceptable salt, preferably an acid addition salt formed by apharmaceutically acceptable free acid. The acid addition salt may beobtained from inorganic acids such as hydrochloric acid, nitric acid,phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid,nitrous acid, phosphorous acid, and the like, or non-toxic organic acidssuch as aliphatic monocarboxylate, aliphatic dicarboxylate,phenyl-substituted alkanoate, hydroxyalkanoate, hydroxyalkandioate,aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Thepharmaceutically non-toxic salt may include sulfate, pyrosulfate,bisulfate, sulfite, bisulfate, nitrate, phosphate, monohydrogenphosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride,bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate,acrylate, formate, isobutyrate, caprate, heptanoate, propiolate,oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate,butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate,methybenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate,phthalate, terephthalte, benzene sulfonate, toluene sulfonate, chlorobenzene sulfonate, xylene sulfonate, phenyl acetate, phenyl propionate,phenyl butyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate,tartrate, methane sulfonate, propane sulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate, or mandelate.

The acid addition salt of the present invention may be produced by aconventional method, for example, by dissolving the compound representedby chemical formula 1 in an excess of aqueous acid solution, andprecipitating the salt using a water-miscible organic solvent, forexample, methanol, ethanol, acetone, or acetonitrile.

Also, the compound represented by chemical formula 1 according to thepresent invention may be used in the form of a pharmaceuticallyacceptable metal salt by using a base. For example, an alkaline metal oralkaline earth metal salt may be obtained by dissolving the compoundrepresented by chemical formula 1 in an excess of alkaline metalhydroxide or alkaline earth metal hydroxide solution, filtering off anon-dissolved compound salt, and evaporating and drying the filtrate. Inthis instance, as the metal salt, a sodium salt, a potassium salt, or acalcium salt is suitable in a pharmaceutical aspect. Also, acorresponding silver salt may be obtained by reacting an alkaline metalor alkaline earth metal salt with a suitable silver salt, for example,silver nitrate.

Meanwhile, the composition for preventing and treating influenza virusinfection according to the present invention may be a pharmaceuticalcomposition.

When the composition of the present invention is a pharmaceuticalcomposition, the composition may include a pharmaceutically acceptablecarrier. The composition including the pharmaceutically acceptablecarrier may be variously formulated for oral or parenteraladministration. For preparations, a diluent or an excipient such as afiller, a bulking agent, a binder, a wetting agent, a desintegrant, asurfactant, and the like may be generally added. A solid preparation fororal administration may be used as a tablet, a pellet, powder, agranule, a capsule, and the like, and may be obtained by mixing at leastone compound with at least one excipient, for example, starch, calciumcarbonate, sucrose or lactose, gelatine, and the like. Besides a basicexcipient, a lubricant such as magnesium stearate or talc may be used. Aliquid preparation for oral administration may be used as a suspension,a solution, an emulsion, a syrup, and the like, and may include a commondiluent such as water or liquid paraffin, as well as a variety ofexcipients, for example, a wetting agent, a sweetener, a flavoringagent, a preservative, and the like. A preparation for parenteraladministration may be used as a sterilized aqueous solution, anon-aqueous solvent, a suspension, an emulsion, a lyophilizedpreparation, a suppository, and the like. As a non-aqueous solvent or asuspending agent, propylene glycol, polyethylene glycol, plant oils suchas olive oil, or injectable ester such as ethyl oleate may be used. As asuppository base, witepsol, macrogol, TWEEN® 61, cacao butter, laurinum,or glycerolgelatin may be used.

The pharmaceutical composition may be formulated as at least oneselected from a tablet, a pellet, powder, a granule, a capsule, asuspension, a liquid, an emulsion, a syrup, a sterilized aqueoussolution, a non-aqueous solvent, a lyophilized preparation, and asuppositorie.

The composition of the present invention may be administered in apharmaceutically effective dosage. The term “pharmaceutical effectivedosage” described herein refers to an amount sufficient to treat adisease at a reasonable benefit/danger ratio within the range applicableto a medical treatment, and an effective dosage level may be determinedbased on the type, disease severity, age, and gender of individual, typeof infected virus, drug efficacy, sensitivity to medication, time androute of administration, ration of excretion, period of treatment, drugcombination, and factors well known in the medical field.

However, for preferable effects, a dosage of the turmeric extract or itsfraction according to the present invention may be 0.0001 to 100 mg/kgper day, preferably 0.001 to 100 mg/kg per day, and a dosage of thecompound represented by chemical formula 1 may be 0.0001 to 100 mg/kgper day, preferably 0.001 to 10 mg/kg per day. The composition of thepresent invention may be administered alone or in combination withdrugs, and may be administered sequentially or simultaneously withconventional drugs. Also, the composition may be administered in asingle or multiple dose. Taking all the above factors intoconsideration, it is important to administer such a dosage as to obtaina maximum effect with a minimum amount without side effects, and thedosage may be easily determined by a person having an ordinary skill inthe art.

The composition of the present invention may be used alone or incombination with operation, endocrinotherapy, drug treatment, andmethods using a biological response modifier, to prevent and treatinfluenza virus infection.

Also, the present invention provides a use of a turmeric extract,turmeric fraction, or compound represented by the following chemicalformula 1 in preventing and treating influenza virus infection, or a useof a turmeric extract, turmeric fraction, or compound represented by thefollowing chemical formula 1 in preparing a drug for preventing andtreating influenza virus infection:

Also, the present invention provides a method for treating a diseasecaused by influenza virus infection including administering apharmaceutically effective dosage of the composition for preventing andtreating influenza virus infection to an individual infected or to beinfected with influenza virus. The influenza virus may be influenza Avirus, influenza B virus, or influenza C virus, preferably H1N1influenza virus or H9N2 influenza virus. More preferably, the influenzavirus may be rvH1N1 influenza A virus, H1N1 influenza virus (A/PR/8/34),or H₉N₂ influenza virus (A/Chicken/Korea/MS96/96). The disease caused byinfluenza virus infection may be influenza, cold, laryngopharyngitis,bronchitis, pneumonia, and particularly, bird flu, swine flu, or goatflu.

The term “individual” described herein refers to all animals includinghumans infected or to be infected with influenza virus, and the diseasemay be effectively prevented and treated by administering a compositionincluding the extract, fraction, or compound of the present invention tothe individual. For example, the composition of the present inventionmay treat humans infected with human influenza virus of a variety ofinfluenza virus subtypes or variants.

Also, the composition of the present invention may treat humans infectedwith avian influenza virus of a variety of influenza virus subtypes orvariants. Also, the composition of the present invention may treatchickens or swines infected with avian influenza virus of a variety ofinfluenza virus subtypes or variants.

The composition of the present invention may be administered incombination with a conventional drug used for a disease caused byinfluenza virus infection. For example, the conventional drug mayinclude, but is not limited to, Amatadine, Rimatadine, Zanamivir, andOseltamivir.

The composition of the present invention may be administered via anygeneral route as long as the composition can be delivered to a targettissue. The composition of the present invention may be administered byan intraperitoneal, intravenous, intramuscular, subcutaneous,intradermal, oral, intranasal, intralung, or rectal route ofadministration depending on the intent of administration, however thepresent invention is not limited in this regard. Also, the compositionmay be administered using any device capable of delivering an activeingredient to a target cell.

Further, the present invention provides a food composition comprising aturmeric extract, a turmeric fraction, or a compound represented by thefollowing chemical formula 1 separated therefrom as an active ingredientto prevent and reduce influenza virus infection:

where R is independently hydrogen, a hydroxyl group, or C₁-C₁₀ alkoxygroup.

In this instance, the compound represented by chemical formula 1 may beseparated from the turmeric extract or turmeric fraction. Morespecifically, the compound represented by chemical formula 1 may be acompound represented by any one of the following chemical formulas 2 to4.

In other words, the turmeric extract, the turmeric fraction, or thecompound represented by chemical formula 1 separated therefrom accordingto the present invention may be added to a food composition to preventor reduce influenza virus infection.

According to the present invention, the food composition may be afunctional food, and the “functional food” is a food with a bodymodulating function, and refers to a value added food that works on andis expressed at a specific target using physical, biochemical, andbiotechnological methods. The ingredients of the functional food may bedesigned and processed to sufficiently exert a body control functioninvolving biological defense, biorhythm control, and disease preventionand recovery on the body.

When the turmeric extract, the turmeric fraction, or the compoundrepresented by chemical formula 1 separated therefrom according to thepresent invention is used as a food additive, the extract, fraction, orcompound may be used as it is or may be used together with foods or foodingredients, and may be properly used by a conventional method. A mixratio of active ingredients may be suitably determined depending on thepurpose of use, for example, disease prevention or therapeutictreatment. Generally, when manufacturing foods or beverages, theturmeric extract, the turmeric fraction, or the compound represented bychemical formula 1 separated therefrom according to the presentinvention may be added in an amount of 0.01 to 10 w %, preferably 0.05to 1 w %, based on the raw material composition. In the case of along-term use for health, sanitation or diet, the amount may be lessthan the above range.

The food is not limited to a specific kind. For example, the foodcomprising the above substance may be meats, sausages, breads,chocolates, candies, snacks, cookies, pizza, ramen, noodles, gums, dairyproducts including ice creams, various soups, beverages, teas, healthdrinks, alcoholic beverages, vitamin complexes, and the like, and mayinclude all traditional health foods.

The health beverage composition of the present invention mayadditionally contain a flavoring agent or natural carbohydrate, like ageneral beverage. The natural carbohydrate may be monosaccharide such asglucose and fructose, disaccharide such as maltose and sucrose,polysaccharide such as dextrin and cyclodextrin, or sugar alcohol suchas xylitol, sorbitol and erythritol. The sweetener may be a naturalsweetener such as thaumatin and a stevia extract, or a syntheticsweetener such as saccharin and aspartame. The content of the naturalcarbohydrate may be generally about 0.01 to 0.04 g, preferably about0.02 to 0.03 g, per 100 d of the composition of the present invention.

In addition to the above, the composition of the present invention maycontain a variety of additives, for example, nutrient, vitamin,electrolyte, a flavoring agent, a coloring agent, pectic acid and itssalt, alginic acid and its salt, organic acid, a protective colloidthickening agent, a pH adjusting agent, a stabilizer, a preservative,glycerin, alcohol, a carbonating agent for carbonated beverages, and thelike. The content of the additive is not particularly important, but maybe generally selected within the range of 0.01 to 0.1 parts by weightsper 100 parts by weight of the composition of the present invention.These additives may be used alone or in combination.

Further, the present invention provides a quasi-drug compositioncomprising a turmeric extract, a turmeric fraction, or a compoundrepresented by the following chemical formula 1 separated therefrom asan active ingredient to prevent and reduce influenza virus infection:

where R is independently hydrogen, a hydroxyl group, or C₁-C₁₀ alkoxygroup.

In this instance, the compound represented by chemical formula 1 may beseparated from the turmeric extract or turmeric fraction. Morespecifically, the compound represented by chemical formula 1 may be acompound represented by any one of the following chemical formulas 2 to4.

In other words, the composition of the present invention may be added toa quasi-drug composition to prevent or reduce influenza virus infection.When the turmeric extract, turmeric fraction, compound represented bychemical formula 1 separated therefrom according to the presentinvention is used as a quasi-drug additive, the turmeric extract,fraction, or compound may be used as it is or may be used together withquasi-drugs or quasi-drug ingredients, and may be properly used by aconventional method. A mix ratio of active ingredients may be suitablydetermined depending on the purpose of use, for example, diseaseprevention, health, or therapeutic treatment.

Preferably, the quasi-drug composition may be used in manufacturing anatural disinfectant, a feed additive, a disinfectant cleaner, a showerfoam, a mouthwash, a wet tissue, a detergent soap, a hand-wash, ahumidifier filler, a mask, an ointment, a filter filler, and the like.

Also, the present invention provides a composition comprising a turmericextract, a turmeric fraction, or a compound represented by the followingchemical formula 1 as an active ingredient to inhibit neuraminidaseactivity:

where R is independently hydrogen, a hydroxyl group, or C₁-C₁₀ alkoxygroup.

In this instance, the neuraminidase may be derived from influenza Avirus, influenza B virus, influenza C virus, Clostridium perfringens, orhuman, preferably H1N1 influenza virus or H9N2 influenza virus. Morepreferably, the influenza virus may be derived from rvH1N1 influenza Avirus, H1N1 influenza virus (A/PR/8/34), or H₉N₂ influenza virus(A/Chicken/Korea/MS96/96).

Neuraminidase, also known as sialidase or acylneuraminyl hydrolase, isan enzyme present in animals and some microorganisms, and manymicroorganisms containing neuraminidase may cause diseases to humans andanimals including poultry, horses, swines, seals, and the like.Accordingly, the composition for inhibiting neuraminidase activityaccording to the present invention may be useful in preventing andtreating many diseases related to neuraminidase activity.

Also, the present invention provides a method for inhibitingneuraminidase activity including contacting the composition with asample containing neuraminidase. In this instance, the contacting may becarried out by a conventional method, and the sample containingneuraminidase may be living organisms, tissues or cell cultivators, orbiological samples such as blood, serum, urine, cerebrospinal fluid,tear, sputum, saliva, tissue, and the like, and may includeneuraminidase-producing organisms, generally pathogenic organisms suchas viruses. These samples may be put in any culture medium containingwater, or an organic solvent/water mixture.

After the composition of the present invention is administered, theneuraminidase activity may be observed by any method including direct orindirect diagnosis of the neuraminidase activity. Also, quantitative,qualitative, or semi-quantitative diagnosis of neuraminidase activity,and observation of the physiological characteristics of living organismsmay be used.

DETAILED DESCRIPTION

Hereinafter, the present invention will be described in detail throughexamples and experimental examples. However, the description proposedherein is just a preferable example for the purpose of illustrationsonly, not intended to limit the scope of the invention, so it should beunderstood that the examples are provided for a more definiteexplanation to an ordinary person skilled in the art.

Example 1 Preparation of Turmeric Extract

A turmeric used in this example is generally available in a medicinalherb shop or market. The steamed and dried radix of Curcuma longa Linnewas purchased, and to effectively obtain an extract of the presentinvention, was milled in powder form. 1.6 kg of turmeric was dissolvedin 7.5 l of 100% ethanol (EtOH), placed at room temperature for 5 days,and filtered through a filter paper, to obtain 170 g of a turmericethanol extract.

Example 2 Separation of Turmeric Fraction and Curcuminoid-Based Compoundfrom Turmeric Extract and Purification

170 g of the turmeric ethanol extract obtained in example 1 wassuspended in 1 l of water. A separatory funnel was placed, and thesuspension was fractionally extracted with n-hexane and ethylacetate insequence, to yield 23 g of an n-hexane soluble extract, 85 g of anethylacetate soluble extract, and 34 g of a water soluble extract.

85 g of the obtained ethylacetate soluble extract was separated into 15fractions Fr.-1 to Fr.-15 by silica gel column chromatography (500 gsilica gel, mesh 70˜230) using solvents of chloroform, methanol, andmixture thereof (80:1˜1:1) as mobile phases. Among them, 16 g of thesixth fraction Fr.-6 was separated into 5 fractions Fr.-6-1 to Fr.-6-5by silica gel column chromatography (30 g, mesh 230˜400) using a mixedsolvent of n-hexane and ethylacetate (20:1˜1:1 (v/v)) as a mobile phase.

After performing silica gel column chromatography on Fr.-6-2 and Fr.-6-3fractions (11 g) using solvents of chloroform, methanol, and mixturethereof (80:1˜4:1) as mobile phases, a fraction obtained was developedby preparative TLC using a mixed solvent of n-hexane and ethylacetate(4:1 (v/v)) as a mobile phase, to yield 8 g of a pure compound 1. Also,14 g of the eighth fraction Fr.-8 was repetitively separated by silicagel column chromatography (30 g, mesh 230˜400) using a mixed solvent ofn-hexane and ethylacetate (20:1˜1:1 (v/v)) and a mixed solvent ofchloroform and methanol (80:1˜20:1 (v/v)) as mobile phases, to yield 0.4g of a compound 2 and 0.2 g of a compound 3.

Example 3 Structural Analysis of Curcuminoid-Based Compound

The molecular weight and molecular formula of the curcuminoid-basedcompounds obtained in example 2 was determined using a VG highresolution GC/MS spectrometer (Election Ionization MS, Autospec-Ultima).Also, the molecular structure was determined through nuclear magneticresonance analysis (Bruker AM500) using ¹H-NMR, ¹³C-NMR, and 2D NMRspectroscopy materials.

Based on comparing the above results with those of published papers,curcumin, demethoxycurcumin and bisdemethoxycurcumin represented bychemical formulas 2 to 4 were identified (Food Chem. 265-272, 2009; J.Nat. Prod. 1227-1231, 2002; J. Nat. Prod. 1531-1534, 1998; J. Agric.Food Chem. 3668-3672, 2002). The analysis results are shown in detailbelow.

Compound 1: Curcumin

1) Property: light orange powder (m.p. 183° C.)

2) Molecular weight: 368.3

3) Molecular formula: C₂₁H₂₀O₆

4) ¹H-NMR (acetone-d₆, 500 MHz) δ 7.62 (2H, d, J=15.80 Hz, H-4, H-4′),7.35 (2H, d, J=1.91 Hz, H-6, H-6′), 6.83 (2H, H-3, H-5), 7.20 (2H, dd,J=8.3, 1.9 Hz, H-10, H-10′), 6.90 (2H, d, J=8.15 Hz, H-9, H-6′), 5.99(1H, s, H-1), ¹³C-NMR (acetone-d₆, 125 MHz) δ 56.72, 102.01, 111.95,116.64, 122.72, 124.25, 128.58, 141.81, 149.20, 150.44, 184.94.

Compound 2: Demethoxycurcumin

1) Property: orange powder (m.p. 220° C.)

2) Molecular weight: 338

3) Molecular formula: C₂₀H₁₈O₅

4) ¹H-NMR (acetone-d₆, 500 MHz) δ 7.62-7.55 (4H), 7.34 (1H), 7.18 (1H),6.89 (3H), 6.70 (2H), 5.97 (1H), ¹³C-NMR (acetone-d₆, 125 MHz) δ 56.38,101.79, 111.53, 116.30, 116.89, 122.12, 122.35, 123.98, 127.77, 128.24,131.06, 141.13, 141.48, 148.87, 150.13, 160.64, 184, 66.

Compound 3: Bisdemethoxycurcumin

1) Property: orange powder (m.p. 224° C.).

2) Molecular weight: 308

3) Molecular formula: C₁₉H₁₆O₄

4) ¹H-NMR (acetone-d₆, 500 MHz) δ 7.62-7.56 (6H), 6.91-6.87 (4H),6.68-6.65 (2H), 5.98 (1H), ¹³C-NMR (acetone-d₆, 125 MHz) δ 101.82,116.87, 122.11, 127.79, 131.06, 141.12, 160.58, 184, 62.

Example 4 HPLC Analysis of Turmeric Alcohol Extract and Fraction

The turmeric extract, the turmeric fraction, and the curcuminoid-basedcompounds obtained in examples 1 and 2 were analyzed by HPLCchromatography. In this example, Agilent 1200 series HPLC system with apump and a variable wavelength detector (VWD) was used. In thisinstance, wavelength was 260 nm for optimum observation, and a flow rateof a solvent was 1.0 ml/min. In the sample preparation, an injectionvolume was 10 μl and a sample concentration was 10 mg/ml. In the HPLCanalysis, ZORBAX-SB-18 column (5 μm, 150 mm×4.6 mm) was used. 20%acetonitrile (0 min), 25% acetonitrile (10 min), 35% acetonitrile (20min), 50% acetonitrile (30 min), 60% acetonitrile (40 min), 70%acetonitrile (50 min), and 100% acetonitrile (60 min) were used as amobile phase on the condition of polar distribution of water (containing0.1% TFA) and acetonitrile (containing 0.1% TFA).

In this example, to measure the content of the curcuminoid-basedcompounds contained in each of the turmeric extract and the turmericfraction, calibration curves were prepared under the same conditions asthe above at 15.625, 31.25, 62.5, 125, 250, 500, and 1000 μg/ml ofcurcumin, demethoxycurcumin, and bisdemethoxycurcumin.

Based on the black line, the measurement results of the content ofcurcumin, demethoxycurcumin, and bisdemethoxycurcumin in each of theturmeric extract and the turmeric fraction showed that 106.0 g/kg ofcurcumin (extract), 45.4 g/kg of demethoxycurcumin (extract), and 64.6g/kg of bisdemethoxycurcumin (extract) were contained in the turmericethanol extract, 114.3 g/kg of curcumin (fraction), 45.8 g/kg ofdemethoxycurcumin (fraction), and 66.4 g/kg of bisdemethoxycurcumin(fraction) were contained in the ethylacetate fraction, and 21.3 g/kg ofcurcumin (fraction), 7.3 g/kg of demethoxycurcumin (fraction), and 31.2g/kg of bisdemethoxycurcumin (fraction) were contained in the hexanefraction.

The quantitative results are shown in Table 1 below.

TABLE 1 Content Sample used Curcumin DemethoxycurcuminBisdemethoxycurcumin Turmeric ethanol 106.0 g/kg (Extract) 45.4 g/kg(Extract) 64.6 g/kg (Extract) extract Turmeric ethylacetate 114.3 g/kg(Fraction) 45.8 g/kg (Fraction) 66.4 g/kg (Fraction) fraction Turmerichexane  21.3 g/kg (Fraction)  7.3 g/kg (Fraction) 31.2 g/kg (Fraction)fraction

Further, by using the black line calculation, the measurement results ofthe content of curcumin, demethoxycurcumin, and bisdemethoxycurcumin inan alcohol extract of Curcuma longa Linne for area showed that 17.0 g/kgof curcumin (extract), 5.3 g/kg of demethoxycurcumin (extract), and 3.4g/kg of bisdemethoxycurcumin (extract) were detected in a tuberous root,curcumin, demethoxycurcumin, and bisdemethoxycurcumin were not detectedin a stem, and 2.8 g/kg of curcumin (fraction), 0.4 g/kg ofdemethoxycurcumin (fraction), and 6.8 g/kg of bisdemethoxycurcumin(fraction) were detected in a rhizome.

The quantitative results are shown in Table 2 below.

TABLE 2 Sample Content (g/Kg, Content) used Curcumin DemethoxycurcuminBisdemethoxycurcumin Ethanol 17.0 6.3 3.4 extract of tuberous rootEthanol — — — extract of stem Ethanol  2.8 0.4 6.8 extract of rhizome

Experimental Example 1 Determination of NeuraminidaseA/Bervig_Mission/1/18 (rvH1N1) Inhibitory Activity of Turmeric Extract,Fraction, and Curcuminoid-Based Compound

To determine the neuraminidase inhibitory activity of the turmericextract, the turmeric fraction, and the curcuminoid-based compoundsseparated therefrom that were obtained in examples 1 and 2 of thepresent invention, neuraminidase (R&D SYSTEM, 4858-NM) of a recombinantrvH1N1 influenza A virus of 1918 Spanish flu virus(A/Bervig_Mission/1/18) was used.2′-(4-trimethylumbelliferyl)-α-D-N-acetyl-neuraminic acid sodium saltfrom Sigma was used as a substrate.

The turmeric extract and its fraction of examples 1 and 2 were dissolvedin methanol. As a substrate,2′-(4-trimethylumbelliferyl)-α-D-N-acetyl-neuraminic acid sodium salt(final concentration 200 μM) was added to 20 μL of each solution. Theresult was mixed with 80 μL of a tris buffer (pH 7.5) containing 5 mMCaCl₂ and 200 mM NaCl, and reacted with 50 μL of neuraminidase (finalenzyme concentration 0.05 ng/μL) as a zymogen at 25° C. for 10 minutes.The neuraminidase inhibitory activity was determined by measuring thelight absorption at 365 nm and the light emission at 445 nm using afluorescence spectroscope.

The measurement results are shown in Table 3 below.

TABLE 3 Neuraminidase inhibitory activity (IC₅₀)¹⁾ A/Bervig-Mission/Substance 1/18 (rvH1N1) Turmeric ethanol extract 3.1 μg/mL Turmerichexane fraction 262.0 μg/mL Turmeric ethylacetate fraction 0.9 μg/mLTurmeric water fraction 61.5 μg/mL Curcumin 3.0 μM Demethoxycurcumin 3.0μM Bisdemethoxycurcumin 6.0 μM [Note] ¹⁾a result value is an average oftwo tests.

As shown in Table 3, the determination results of neuraminidaseinhibitory activity of each of the turmeric extract, fraction, andcurcuminoid-based compounds according to the present invention showedthat the turmeric ethanol extract had an IC₅₀ value of 3.1 μg/mL againstinfluenza virus neuraminidase, the turmeric hexane fraction had an IC₅₀value of 262.0 μg/mL, the turmeric ethylacetate fraction had an IC₅₀value of 0.9 μg/mL, the turmeric water fraction had an IC₅₀ value of61.5 μg/mL, curcumin and demethoxycurcumin had each an IC₅₀ value of 3.0μM, and bisdemethoxycurcumin had an IC₅₀ value of 6.0 μM. Accordingly,it was determined through the above results that the turmeric extract,its fraction, and curcuminoid-based compounds according to the presentinvention had excellent neuraminidase inhibitory activity.

Experimental Example 2 Determination of Influenza Virus InhibitoryActivity of Curcuminoid-Based Compound Separated from Turmeric Extract,Ethylacetate Fraction, and Ethanol Extract

To determine the antiviral effects of the curcuminoid-based compounds,ethylacetate fraction, and ethanol extract separated from the turmericextract on influenza virus H1N1 (A/PR/8/34) and H₉N₂(A/Chicken/Korea/MS96/96), a test below was carried out in vitro using aMadin-Darby canine kidney (MDCK, ATCC CCL-34) cell of a dog.

First, MDCK cells were put in a 96-well microplate at a density of1×10⁵/well, and incubated in a culture medium (EMEM) containing 100units penicillin, 100 μg streptomycin and 10% FBS. When the MDCK cellsare grown as a monolayer, the MDCK cells were washed twice with aculture medium (EMEM) only containing antibiotic. Each of H1N1 and H9N2strains was diluted with 100 TCID₅₀, and put in an EP tube. Thecurcuminoid-based compounds, ethylacetate fraction and ethanol extractseparated from the turmeric extract diluted with dimethylsulfoxide(DMSO) were put in each tube based on concentration, followed byreaction at 4° C. for 1 hour. After the lapse of 1 hour, the reactedsolutions were inoculated into 3 wells of the pre-washed MDCK cells ateach concentration, followed by incubation at 35° C. for 1 hour(hereinafter referred to as a test sample). Under the same conditions,non-infected+non-administered cells (MDCK cells not infected with anH1N1 or H9N2 strain and not administered with the curcuminoid-basedcompound) and infected+non-administered cells (MDCK cells infected withan H1N1 or H9N2 strain and not administered with curcumin), incubated at35° C. for 1 hour, were each set as a control and a virus control. Afterthe lapse of 1 hour, the culture mediums of the plate were all removed,and the cells were washed once with PBS. 10 μg/mL of a culture medium(EMEM) containing antibiotic and 10 μg/mL trypsin was dispensed in eachwell of the cells, followed by incubation at 35° C. for 48 to 72 hours.The incubation was performed for 48 to 72 hours until theinfected+non-administered cells (virus control) have cytopathic effects(CPE). The state of the cells was observed with an inverted microscopeevery day. After the cells were incubated for 48 to 72 hours, 10 ml of acell counting kit-8 (Dojin, Kumanoto, Japan, tetrazolium salt WST-8) wasadded to each well to determine the cell survival, followed by reactionat 35° C. for 2 hours, and the absorbance was measured at 450 nm. Inthis instance, the antiviral effects (inhibition (%)) of thecurcuminoid-based compounds, ethylacetate fraction, and ethanol extractseparated from the turmeric extract of the present invention werecalculated using the following mathematical formula 1 in comparison withthe non-infected+non-administered cells (control) and theinfected+non-administered cells (virus control). The results are shownin Table 4 below.

                               Mathematical  formula  1${{Inhibition}(\%)} = \frac{\begin{matrix}{{O\; D\mspace{14mu} {value}\mspace{14mu} {of}\mspace{14mu} {test}\mspace{14mu} {sample}} - {O\; D\mspace{14mu} {value}\mspace{14mu} {of}\mspace{14mu} {infected}} +} \\{{non}\text{-}{administered}\mspace{14mu} {control}}\end{matrix}}{\begin{matrix}{{O\; D\mspace{14mu} {value}\mspace{14mu} {of}\mspace{14mu} {non}\text{-}{infected}} + {{non}\text{-}{administered}\mspace{14mu} {control}} -} \\{{O\; D\mspace{14mu} {value}\mspace{14mu} {of}\mspace{14mu} {infected}} + {{non}\text{-}{administered}\mspace{14mu} {control}}}\end{matrix}}$

where the OD value is absorbance measured at 450 nm.

Meanwhile, to determine the cell degeneration inhibitory effects of thecurcuminoid-based compounds, ethylacetate fraction, and ethanol extractseparated from the turmeric extract on influenza virus H1N1 (A/PR/8/34)and H9N2 (A/Chicken/Korea/MS96/96), MDCK cells were washed twice with aculture medium (EMEM) only containing antibiotic, inoculated withinfluenza virus (H1N1 or H9N2 strain), and incubated at 35° C. for 1hour. After the lapse of 1 hour, the virus solution used in inoculationwas completely removed, and the curcuminoid-based compounds,ethylacetate fraction, and ethanol extract separated from the turmericextract were each inserted into the virus-inoculated MDCK cells.Subsequently, the cell degeneration inhibitory effects (inhibition (%))of the curcuminoid-based compounds, ethylacetate fraction, and ethanolextract separated from the turmeric extract of the present inventionwere determined using the above mathematical formula 1. The results areshown in Table 4 below.

TABLE 4 Antiviral effect H1N1(A/PR/8/34) H9N2(A/Chicken/Korea/MS96/96)Substance CC₅₀(μM)^(a) EC₅₀(μM)^(b) SI^(c) CC₅₀(μM)^(a) EC₅₀(μM)^(b)SI^(c) Tamiflu >200 18.5 >10.8 >200 <1 >200 Curcumin 94.1 7.1 13.3 94.118.6 5.1 Demethoxycurcumin 97.0 8.0 23.1 97.0 18.2 5.3Bisdemethoxycurcumin >200 28.1 >7.1 >200 >200 <1 Ethylacetate fraction82.1 μg/mL 9.0 μg/mL 9.1 82.1 μg/mL 20.0 μg/mL 4.1 Ethanol extract 55.7μg/mL 8.7 μg/mL 6.4 55.7 μg/mL 30.4 μg/mL 1.8 Cell degenerationinhibitory effect H1N1(A/PR/8/34) H9N2(A/Chicken/Korea/MS96/96)Substance CC₅₀(μM)^(a) EC₅₀(μM)^(b) SI^(c) CC₅₀(μM)^(a) EC₅₀(μM)^(b)SI^(c) Tamiflu >200 3.0 >66.7 >200 <1 >200 Curcumin 94.1 40.7 2.3 94.110.9 8.6 Demethoxycurcumin 97.0 60.8 1.6 97.0 24.9 3.9Bisdemethoxycurcumin >200 141.5 >1.4 >200 181.6 >1.1 Ethylacetatefraction 82.1 μg/mL 23.5 μg/mL 3.5 82.1 μg/mL 11.6 μg/mL 7.1 Ethanolextract 55.7 μg/mL 27.8 μg/mL 2.0 55.7 μg/mL 31.5 μg/mL 1.8 [Note]^(a)CC₅₀, 50% cytotoxic concentration ^(b)EC₅₀, 50% antiviralconcentration ^(c)SI, selective index, CC₅₀/EC₅₀

a. The curcuminoid-based compounds, ethylacetate fraction and ethanolextract were each mixed with virus, followed by reaction at 4° C. for 1hour, and MDCK cells were infected with the virus. After the lapse of 1hour, the cells were washed once with PBS and a culture medium (EMEM)containing 10 mg/mL trypsin was dispensed. The cells were incubated at35° C. for 48 to 72 hours.

b. After 1 hour of the virus infection, the medium including the viruswas removed and replaced with a fresh medium containing each of thecurcuminoid-based compounds, ethylacetate fraction, and ethanol extract,and then the cells were incubated for 48 to 72 hours.

c. CC₅₀/EC₅₀ as selective index (SI)

As shown in Table 4, the curcuminoid-based compounds had excellentantiviral effects on an H1N1 strain exhibiting a selective index (SI) of13.3, 23.1, and greater than 7.1. Also, the curcuminoid-based compoundsexhibited a selective index (SI) of 5.1, 5.3, and less than 1.0 againstan H9N2 strain. Accordingly, it was determined that thecurcuminoid-based compounds had antiviral effects on a variety of virusstrains. The turmeric ethylacetate fraction and turmeric ethanol extracthad excellent antiviral effects on an H1N1 strain, exhibiting aselective index (SI) of 9.1 and 6.4, respectively. According to theobservation results of the cell morphology with an inverted microscope,it was found that MDCK cells inoculated with virus (H1N1 or H9N2) werealmost degenerated, leading to 90 to 100% of cytopathic effects, whilevirus-infected MDCK cells treated with the curcuminoid-based compounds,ethylacetate fraction, and ethanol extract exhibited a similar aspect toa non-infected+non-administered control.

Furthermore, the curcuminoid-based compounds had cell degenerationinhibitory effects on H1N1 and H9N2. In particular, thecurcuminoid-based compounds had excellent cell degeneration inhibitoryeffects on an H9N2 strain exhibiting a selective index (SI) of 8.6, 3.9,and greater than 1.1, and had cell degeneration inhibitory effects on anH1N1 strain exhibiting a selective index (SI) of 2.3, 1.6, and greaterthan 1.4. According to the observation results of the cell morphologywith an inverted microscope, it was found that MDCK cells inoculatedwith virus (H1N1 or H9N2) were almost degenerated, leading to 90 to 100%of cytopathic effects, while virus-infected MDCK cells treated with thecurcuminoid-based compounds, ethylacetate fraction, and ethanol extractexhibited a similar aspect to a non-infected+non-administered control.

Accordingly, the composition of the present invention may be useful inpreventing and treating influenza virus infection because thecomposition has antiviral effects by directly working on the virusbefore the virus infects cells, and has excellent cell degenerationinhibitory effects by preventing the virus from releasing from cellsthrough replication after the cells are infected with the virus.

Experimental Example 3 Acute Toxicity Test of Compound of the PresentInvention

To determine the acute toxicity of the compounds of the presentinvention, a test below was performed.

Specific pathogens free (SPF) C57BL/6J mice including 12 female and 12male, aged 6 weeks (sample), were grouped into four groups (3 femalemice and 3 male mice as a test group), and bred in an animal room undertemperature of 22±3° C., humidity of 55±10%, and illumination of12L/12D. Before the test, the mice were gone through domestication for 1week. The mice were allowed to freely eat laboratory animal feeds (feedfor mice and rats, CJ Corporation in Seoul, Republic of Korea) andsterile water.

A sample was prepared by adding 0.5% of TWEEN®80 to each of thecompounds represented by chemical formulas 2 to 4 obtained in example 2at a concentration of 50 mg/mL, and administered in a dosage of 0.04 mL(100 mg/kg), 0.2 mL (500 mg/kg), and 0.4 mL (1,000 mg/kg) per 20 g ofmouse weight. The sample was orally administered in a single dose, andafter administration, side effect or survival was observed for 7 days.That is, on the day of administration, the general symptomatic changeand survival were observed after 1 hour, 4 hours, 8 hours, and 12 hoursof administration, and from the next day of administration to theseventh day, were observed once or more each in the forenoon andafternoon every day.

The acute toxicity test results showed that all the mice administeredwith the sample did not have a noteworthy clinical sign and there was nodead mouse. Also, the toxicity change was not observed in the weightchange, blood test, biochemical examination of blood, and autopsyfindings.

The compounds of the present invention did not show the toxicity changein all the mice until a dosage is 1,000 mg/kg, and accordingly, it wasidentified that the compounds of the present invention are a stablesubstance having 1,000 mg/kg or more of an oral LD₅₀, that is, a minimumlethal dose.

Hereinafter, manufacturing examples of a pharmaceutical preparation anda health food comprising a turmeric extract, a turmeric fraction, acurcuminoid-based compound separated therefrom, or its pharmaceuticallyacceptable salt are described.

Manufacturing Example 1 Manufacture of Pharmaceutical Preparation

1-1. Powder

2 g of a turmeric extract, fraction, compound separated therefrom, orits salt

1 g of lactose

These ingredients were mixed and packed in an air-tight container tomanufacture a pharmaceutical preparation in powder form.

1-2. Tablet

100 mg of a turmeric extract, fraction, compound separated therefrom, orits salt

100 mg of corn starch

100 mg of lactose

2 mg of magnesium stearate

These ingredients were mixed and compressed by a conventional tabletmanufacturing method to manufacture a pharmaceutical preparation intablet form.

1-3. Capsule

100 mg of a turmeric extract, fraction, compound separated therefrom, orits salt

100 mg of corn starch

100 mg of lactose

2 mg of magnesium stearate

These ingredients were mixed and enclosed in a gelatin capsule by aconventional capsule manufacturing method to manufacture apharmaceutical preparation in capsule form.

1-4. Injection

10 μg/ml of a turmeric extract, fraction, compound separated therefrom,or its salt

Dilute hydrochloric acid BP to adjust pH to 3.5

Maximum 1 ml of sodium chloride injection BP

A turmeric extract, fraction, compound separated therefrom, or its saltwas dissolved in a proper volume of sodium chloride injection BP. Dilutehydrochloric acid BP for adjusting the pH to 3.5 and sodium chlorideinjection BP for adjusting the volume were sufficiently mixed with theresulting solution. 5 ml type I clear glass ampoule was filled with thesolution, sealed by fusing the glass under an upper grid of air, andsterilized by autoclaving at 120° C. for 15 minutes or more, tomanufacture a pharmaceutical preparation in injection form.

Manufacturing Example 2 Manufacture of Health Food

2-1. Cooking Condiment

0.2 to 10 w % of a turmeric extract, fraction, compound separatedtherefrom, or its salt was used in manufacturing cooking condiments forhealth promotion.

2-2. Tomato Ketchup and Sauce

0.2 to 1.0 w % of a turmeric extract, fraction, compound separatedtherefrom, or its salt was added to tomato ketchups or sauces tomanufacture tomato ketchups or sauces for health promotion.

2-3. Flour Food

0.1 to 5.0 w % of a turmeric extract, fraction, compound separatedtherefrom or its salt was mixed with flour, and the mixture was used inmaking breads, cakes, cookies, crackers, and noodles, to manufacturefoods for health promotion.

2-4. Soup and Gravy

0.1 to 1.0 w % of a turmeric extract, fraction, compound separatedtherefrom or its salt was added to soups and gravies to manufactureprocessed meat products, soups for noodles, and gravies for healthpromotion.

2-5. Ground Beef

10 w % of a turmeric extract, fraction, compound separated therefrom orits salt was added to ground beef to manufacture ground beef for healthpromotion.

2-6. Dairy Product

0.1 to 1.0 w % of a turmeric extract, fraction, compound separatedtherefrom or its salt was added to milk, and the milk was used inmanufacturing various dairy foods including butter and ice-cream.

2-7. Cereal Powder

Unpolished rice, barley, glutinous rice, and adlay were pregelatinizedby a known method, dried, roasted, and milled into powder having aparticle size of 60 mesh using a mill.

Black soybean, black sesame, and perilla were steamed by a known method,dried, roasted, and milled into powder having a particle size of 60 meshusing a mill.

A turmeric extract, fraction, compound separated therefrom or its saltwas evaporated under reduced pressure using a vacuum evaporator,sprayed, hot-air dried, and milled into dried powder having a particlesize of 60 mesh using a mill.

The obtained cereals, seeds, and dried powder of the turmeric extract,fraction, compound separated therefrom or its salt were mixed at thefollowing ratio.

75 w % of cereals including 35 w % of unpolished rice, 15 w % of adlay,and 25 w % of barley

23 w % of seeds including 7 w % of perilla, 9 w % of black soybean, and7 w % of black sesame

1 w % of turmeric extract, fraction, compound separated therefrom or itssalt

0.5 w % of lingzhi mushroom

0.5 w % of rehmanni

2-8. Carbonated Beverage

5 to 10 w % of sugar, 0.05 to 0.3 w % of citric acid, 0.005 to 0.02 w %of caramel, and 0.01 to 1 w % of vitamin C were mixed. Purified waterwas added to make the total composition 100 w %, yielding a syrup. Thesyrup was sterilized at 85 to 98° C. for 20 to 180 seconds, and mixedwith a coolant at a ratio by volume of 1:4, to prepare a beveragecomposition. 0.5 to 0.82 volume % of carbonic acid gas was injected intothe beverage composition to manufacture carbonated beverages comprisinga turmeric extract, fraction, compound separated therefrom or its salt.

2-9. Health Beverage

1 w % of a turmeric extract, fraction, compound separated therefrom orits salt was uniformly mixed with an additive including 0.5 w % of highfructose corn syrup, 2 w % of oligosaccharide, 2 w % of sugar, 0.5 w %of common salt, and 94 w % of water, then flash-pasteurized, and packedin a small packing container such as a vial, a PET bottle, and the like,to manufacture health beverages.

2-10. Vegetable Juice

0.5 g of a turmeric extract, fraction, compound separated therefrom orits salt was added to 1,000 ml of tomato or carrot juices to manufacturevegetable juices for health promotion.

2-11. Fruit Juice

0.1 g of a turmeric extract, fraction, compound separated therefrom orits salt was added to 1,000 ml of apple or grape juices to manufacturefruit juices for health promotion.

1. A composition comprising a turmeric extract or its fraction as anactive ingredient to prevent and treat influenza virus infection.
 2. Thecomposition according to claim 1, wherein the turmeric is Curcumawenyujin (Y. H. Chen et C. Ling), Curcuma longa Linne, Curcuma longaSalisb., Curcuma zedoaria, Curcuma kwansiensis (S. G. Lee et C. F.Liang), Curcuma aeruginosa, Curcuma phaeocaulis Val. (Zingiberaceae), orCurcuma domestica.
 3. The composition according to claim 1, wherein theturmeric is a tuberous root.
 4. The composition according to claim 1,wherein the extract is obtained with a solvent of water, C₁-C₄ alcohol,or mixtures thereof.
 5. The composition according to claim 1, whereinthe fraction is a hexane fraction, an ethylacetate fraction, or a waterfraction.
 6. The composition according to claim 1, wherein the influenzavirus is influenza A virus, influenza B virus, or influenza C virus. 7.The composition according to claim 1, wherein the influenza virus isH1N1 influenza virus or H9N2 influenza virus.
 8. The compositionaccording to claim 1, wherein the influenza virus is rvH1N1 influenza Avirus, H1N1 influenza virus (A/PR/8/34), or H9N2 influenza virus(A/Chicken/Korea/MS96/96).
 9. The composition according to claim 1,wherein a disease caused by the influenza virus infection is influenza,cold, laryngopharyngitis, bronchitis, pneumonia, bird flu, swine flu, orgoat flu.
 10. The composition according to claim 1, wherein thecomposition is a pharmaceutical composition, a food composition, or aquasi-drug composition.
 11. The composition according to claim 10,wherein the pharmaceutical composition is formulated as at least oneselected from the group consisting of a tablet, a pellet, powder, agranule, a capsule, a suspension, a liquid, an emulsion, a syrup, asterilized aqueous solution, a non-aqueous solvent, a lyophilizedpreparation, and a suppository.
 12. The composition according to claim10, wherein the food composition is at least one selected from the groupconsisting of meats, sausages, breads, chocolates, candies, snacks,cookies, pizza, noodles, gums, ice creams, soups, beverages, teas,health drinks, alcoholic beverages, and vitamin complexes.
 13. Thecomposition according to claim 10, wherein the quasi-drug composition isused in manufacturing at least one selected from the group consisting ofa natural disinfectant, a feed additive, a disinfectant cleaner, ashower foam, a mouthwash, a wet tissue, a detergent soap, a hand-wash, ahumidifier filler, a mask, an ointment, and a filter filler.
 14. Acomposition comprising a compound represented by the following chemicalformula 1 as an active ingredient to prevent and treat influenza virusinfection:

where R is independently hydrogen, a hydroxyl group, or C₁-C₁₀ alkoxygroup.
 15. The composition according to claim 14, wherein the compoundrepresented by chemical formula 1 is a compound represented by any oneof the following chemical formulas 2 to 4:


16. The composition according to claim 14, wherein the influenza virusis influenza A virus, influenza B virus, or influenza C virus.
 17. Thecomposition according to claim 14, wherein the influenza virus is H1N1influenza virus or H9N2 influenza virus.
 18. The composition accordingto claim 14, wherein a disease caused by the influenza virus infectionis influenza, cold, laryngopharyngitis, bronchitis, pneumonia, bird flu,swine flu, or goat flu.
 19. The composition according to claim 14,wherein the composition is a pharmaceutical composition, a foodcomposition, or a quasi-drug composition.
 20. The composition accordingto claim 19, wherein the pharmaceutical composition is formulated as atleast one selected from the group consisting of a tablet, a pellet,powder, a granule, a capsule, a suspension, a liquid, an emulsion, asyrup, a sterilized aqueous solution, a non-aqueous solvent, alyophilized preparation, and a suppository.
 21. The compositionaccording to claim 19, wherein the food composition is at least oneselected from the group consisting of meats, sausages, breads,chocolates, candies, snacks, cookies, pizza, noodles, gums, ice creams,soups, beverages, teas, health drinks, alcoholic beverages, and vitamincomplexes.
 22. The composition according to claim 19, wherein thequasi-drug composition is used in manufacturing at least one selectedfrom the group consisting of a natural disinfectant, a feed additive, adisinfectant cleaner, a shower foam, a mouthwash, a wet tissue, adetergent soap, a hand-wash, a humidifier filler, a mask, an ointment,and a filter filler.
 23. A composition comprising a turmeric extract, aturmeric fraction, or a compound represented by the following chemicalformula 1 as an active ingredient to inhibit neuraminidase activity:

where R is independently hydrogen, a hydroxyl group, or C₁-C₁₀ alkoxygroup.
 24. The composition according to claim 23, wherein theneuraminidase is derived from influenza A virus, influenza B virus,influenza C virus, Clostridium perfringens, or human.
 25. Thecomposition according to claim 23, wherein the neuraminidase is derivedfrom H1N1 influenza virus or H9N2 influenza virus.
 26. A method fortreating influenza virus infection, the method comprising:administering, to an individual infected or to be infected withinfluenza virus, a composition comprising a turmeric extract, a turmericfraction, or a compound represented by the following chemical formula 1as an active ingredient to prevent and treat influenza virus infection:


27. A method for inhibiting neuraminidase activity, the methodcomprising: contacting a composition comprising a turmeric extract, aturmeric fraction, or a compound represented by the following chemicalformula 1 as an active ingredient to prevent and treat influenza virusinfection with a sample comprising neuraminidase:


28. A method for separating a compound represented by the followingchemical formula 1 from a turmeric, the method comprising: extracting aturmeric with a solvent of water, C₁-C₄ alcohol, or mixtures thereof toobtain a turmeric extract; obtaining a hexane fraction, an ethylacetatefraction, or a water fraction from the extract; and separating andpurifying the fraction:


29. The method according to claim 26, wherein the compound representedby chemical formula 1 is a compound represented by any one of thefollowing chemical formulas 2 to 4:


30. A method for preparing a composition of claim 1 having activityagainst neuraminidase, the method comprising: extracting a turmeric witha solvent of water, C₁-C₄ alcohol, or mixtures thereof to obtain aturmeric extract.
 31. The method according to claim 30, furthercomprising: fractioning the turmeric extract with hexane, ethylacetate,or water.
 32. The method according to claim 31, wherein the methodincludes separating and purifying the turmeric fraction.